We reported previously that a biphasic LH release (within 30 min and 90-150 min after LH-RH injection) was observed in normal menstruating baboons (non-human primates). Plasma immunoassayable LH-RH reached a maximum within 4 min after injection and was undetectable within 60 min. Plasma estrogen and progestin were elevated within 45 min. No plasma LH release with increment of plasma estrogen and progestin was observed in saline injected baboons. This study was performed to investigate the mechanism of biphasic LH release by LH-RH injection.
Synthetic LH-RH (100μg) was injected sc into four female retrochiasmatic deafferented baboons. Blood samples collected 30 and 2 min before injection and 5, 10, 15, 30, 45, 60, 90,120,150 and 180 min after injection were assayed for LH, estrogen and progestin. In four baboons plasma LH peak was observed within 30 min after injection and plasma estrogen and progestin were elevated within 45 min. However, plasma LH peak within 90-150 min was not observed. These results infer that exogenous LH-RH exerts an effect on the pituitary to release LH within 30 min after injection. Increased estrogen and progestin exert the effect on the higher brain area (extrahypothalamic area) and the anterior hypothalamus to facilitate release of endogenous LH-RH, which subsequently release LH within 90-150 min in cooperation with exogenous LH-RH which may be considered to participate LH synthesis in the pituitary. Thus, it seems likely that the extrahypothalamic area (for example; limbic system) and the anterior hypothalamus have an important role in regulating LH-RH secretion and subsequent LH release in the baboon.

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A study of the contributions of human pituitary thyrotropin and human chorionic thyrotropin to normal thyroid homeostasis during pregnancy.
With the use of a recently developed, sensitive and specific radioimmunoassay for hCT and hTSH, the author examined the serum concentrations of hCT and hTSH in 220 normal pregnant women from the 2nd month of pregnancy through parturition, and compared the levels with the concentrations of other polypeptide hormones obtained from the same serum sample. To assess the effect of TRH on the secretion of hCT and hTSH, TRH was administrated in a dose of 100pg to 60 pregnant women in the first to the 3rd trimester of gestation. Simultaneously, thyroid function was evaluated with various routine examinations.
Serum hCT was detectable even at the end of the 2nd gestational month (3.3μg/ml ± 1.4 S.E.), tending to increase gradually toward the 3rd trimester (28.0μg/ml±24.6 S.E. at the term). Baseline hTSH values did not change during pregnancy and there were no significant differences in the baseline values in normal non-pregnant (2.7μU/ml ± 1.6 S.E.) and pregnant women (3.9μU/ml ± 2.6 S.E.). Serum hTSH levels increased in all subjects during each trimester after TRH administration. The mean values for ΔhTSH were 13.9μU/ml ± 2.2 S.E. in the first, 14.5μU/ml± 1.9 S.E. in the second and 13.0μU/ml ± 1.5 S.E. in the third trimester. The hTSH response to TRH remained unaltered as compared with non-pregnant women. Serum hCT levels showed no significant change after TRH. Serum T3, T4 concentrations and values of PBI or TBG-binding capacity were demonstrated to rise gradually during pregnancy in a manner similar to serum hCT levels. The mean values for Triosorb decreased gradually and the values of ETR remained within the non-pregnant range throughout pregnancy. And there existed positive correlations not between hTSH and T3, T4 concentrations but between hCT and T3, T4 concentrations in individual samples.
In summary, in non-pregnant women thyroid function is controlled by pituitary hTSH (hypothalamic-pituitary-thyroid axis). In pregnant women, it is possible to say that thyroid function is controlled slightly by hTSH, but is never enhanced by hTSH. Thyroid function in pregnant women is enhanced by hCT itself which has a higher activity in serum than that of hTSH. Meanwhile, TBG-binding capacity is known to be increased by estrogen. Then, estrogen increased during pregnancy makes TBG bind with more thyroid hormones and keeps free thyroxine in the non-pregnant range. TBG neither elevated the serum hTSH level nor altered thyroid function progressively toward the 3rd trimester of pregnancy.
As mentioned above, it may be possible to say that the thyroidal homeostasis during pregnancy is mainly controlled by “placento-thyroidal axis”.

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The leucocyte migration inhibition test (LMT) using the agarose plate method introduced by Clausen is simple and highly reproducible. Using human pancreas extract and beef insulin as antigen, LMT was performed on ten patients with insulin dependent diabetes, twenty patients with insulin independent diabetes, and twelve healthy controls.
The migration index was expressed as a percentage of migration calculated from the following formula.
Migration index (MI) =average areas of migration in test suspension/average areas of migration in control suspension
Using human pancreas extract, the mean migration index for the insulin dependent diabetics (87.6 ± 11.1) was significantly lower than in the normal subjects (99.3 ± 6.3) (p<0.05)
Using beef insulin as antigen for the insulin dependent diabetics and insulin independent diabetics, the mean migration indices ( ±SD) were 95.8±14.9 and 98.7±12.3 respectively.
The corresponding values for the control group were 98.9 ± 7.8. Cellular hypersensitivity to human pancreas extract was shown in the leucocyte migration inhibition test with insulin dependent diabetics, but a negative result was obtained with beef insulin.

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The purpose of this study is to determine the affinities and the binding radicals of progesterone receptor, and their effect on the formation of the progesterone-receptor complex which binds to chromatin.
The uterine cytosol and the uterine chromatin were obtained from the estrogen primed immature female rabbit. 8S components were obtained after the 5-20% sucrose linear gradient centrifugation of uterine cytosol.
1. A sedimentation profile of the progesterone binding macromolecule showed that ³H-progesterone formed 8S and 5S binding complex, and the ³H-progesterone-8S binding was effectively inhibited by norethindrone or ethynodiol diacetate, resulting in the relative increases of ³H-progesterone-5S binding and free progesterone.
The effect of steroids on progesterone-8S binding was studied by a double reciprocal plot which showed that the following steroids were competitive inhibitors for progesterone 8S binding, and the order of the affinity for progesterone receptor was norethindrone (Ki=2.3×10^-9M) >5α-dihydronorethindrone (Ki=6.0×10^-9 M) > norethindrone acetate (Ki=8.8×10^-9M) > lynestrenol (Ki=9.7×10^-9M) >17α-ethynyl-4-estren-3β, 17β-diol (Ki=5.4×10^-8M) > ethynodiol diacetate (Ki=1.3×10^-7M), when taken from Ki. Ki (inhibitor constant) was calculated by the following equation : Ki=iKd/ (Kp-Kd), where Kd is the dissociation constant, i is the concentration of an inhibitor and Kp is the pretending dissociation constant in the presence of an inhibitor.
These results suggest that the 3-ketone and 17β-hydroxyl groups, and the plane of ring A/B of norethindrone are important to bind with progesterone receptor.
2. The uterine cytosols, preincubated with ³H-progesterone (or ³H-norethindrone) alone or in the presence of cold steroids at 4°C for lh 30 min, were incubated with uterine chromatins at 4°C for 1h 30 min. These chromatins were washed, and the radioactivities in them were counted.
It was indicated that the effect of cold steroids of the estrane series on the formation of progesterone- or norethindrone-receptor complex which binds to the chromatin appeared to be similar in their effect on progesterone- or norethindrone-receptor binding.

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A radioimmunoassay method for the measurement of plasma 18-0H-DOC has been developed. The antiserum against 18-OH-DOC was produced in rabbits immunized with 18-OH-DOC-3-oxime-porcine gammaglobulin. Both 1, 2-³H-18-OH-DOC and Standard 18-OH-DOC were stored in 0.1% pyridine ethanol at -20°C. 1, 2-³H-18-0H-DOC was added to all samples to compensate for procedural losses. Plasma (1-3ml) was extracted with dichloromethane and chromatographied on LH-20 column (1×50cm). The purified extracts were incubated with antiserum at a 1/1000 dilution for 30 minutes at 25°C, and then for 16 hours at 4°C. Saturated ammonium sulfate was used to separate free from bound 18-OH-DOC.
Recovery after extraction was 4.6 ± 7.5 (SD) %. The accuracy and precision of the method were acceptable, and a sensitivity of 5pg per sample enabled the measurement of very low levels of plasma 18-OH-DOC. High specificity of the method was obtained by a high specifity of the antiserum and a good separating ability of Sephadex LH-20 column chromatography.
The mean plasma 18-OH-DOC levels at 8 a.m. were 14.3 ± 5.4ng/100ml in 22 normal subjects. There was a marked increase of plasma 18-0H-DOC by ACTH and a decrease by dexamethasone. Upright position after furosemide administration, dietary sodium restriction and angiotensin II infusion increased moderately plasma 18-OH-DOC.
These results confirm that 18-0H-DOC secretion is regulated primarily by the anterior pituitary, and the renin-angiotensin system plays minor role in 18-OH-DOC secretion. When compared to mineralocorticoid activity of aldosterone or DOC, the 18-OH-DOC level of 14.3ng/100ml and its biological activity would seem that 18-0H-DOC plays a minor role in homeostasis in the normal human body.

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As a succeeding work of our previous investigation, where preovulatory changes of steroidogenesis of the rabbit follicles was studied, steroid hormone formation in vitro by developing corpora lutea isolated from the rabbit has been investigated in the present communication. Five to eight developing corpora lutea following the rupture of preovulatory follicles were isolated from rabbit ovaries under stereomicroscope at the 15th, 18th, 24th, 48th, 72th and 96th hour after intravenous injection of 100IU/kg of human chorionic gonadotropin (hCG) and incubated with 100μCi of acetate-1-¹⁴C in Krebs-Ringer bicarbonate buffer at 37°C for 3 hours under an atmosphere of 95% oxygen plus 5% carbon dioxide. Each incubation was terminated by freezing quickly and stored at - 20°C until twenty to thirty-one corpora lutea had been collected for each time period before commencement of analysis. Incorporations of radioactive acetate into pregnenolone, 17α-hydroxypregnenolone, progesterone, 17α-hydroxyprogesterone, 20a-hydroxyprogesterone, dehydroepiandrosterone, androstenedione, testosterone, estrone and estradiol-17β were analysed by the reverse dilution technique with recrystallization to constant specific activity and expressed as dpm per 20 corpora lutea after correction for analytical losses.
The overall incorporation into the ten steroids remained at the minimal rate observed just prior to ovulation until the 15th hour, began to increase by the 18th hour, and increased constantly toward completion of corpus luteum formation. A comparable quantitative change was found with ¹⁴C incorporation into C₂₁ steroids. However, incorporation into C₁₉ steroids elevated temporarily between the 18th and 48th hours with drastic decrease by the 72 hour.
Incorporation into C₁₈ steroids showed a transient rise at the 18th hour, followed by a gradual decrease to nil by the 72th hour after hCG injection.
Progesterone was the most prominent product formed from acetate-¹⁴C at each point of time in the course of corpus luteum formation. The other major steroidal products varied in the interval between follicle rupture and completion of corpora lutea; i.e. 17α-hydroxy-progesterone and 20α-hydroxyprogesterone at the 15th hour, 17α-hydroxyprogesterone and androstenedione at the 18th hour, 17α-hydroxyprogesterone and 20α-hydroxyprogesterone at the 24th and 48th hours, pregnenolone and 20α-hydroxyprogesterone at the 72th and 96th hours. Distribution patterns of radioactivity among the individual steroids achieved a complete accordance by the 96th hour with that of corpus luteum of pregnant rabbits as previously reported.
It was thus pointed out that marked qualitative as well as quantitative changes in steroidogenesis of developing corpora lutea occurred during the process of corpus luteum formation.

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The role of baroreceptors in common carotid and vertebral arteries and arteries in the thoracic cavity in vasopressin secretion was investigated in this study.
Effects of bilateral occlusion of common carotid and vertebral arteries on blood ADH level as well as mean arterial pressure were studied in common carotid arterial plexus-denervated dogs, cervically vagotomized dogs and intact dogs.
Blood ADH titers were determined by bioassay technic before and 5 minutes after the occlusion of the arteries and were compared with the changes of mean arterial pressure (MAP). The following results were obtained.
(1) Blood ADH titers and MAP were elevated by the occlusion of the common carotid arteries in both intact and vagotomized dogs, while they were not significantly affected in denervated dogs. Elevation of blood ADH titers was more pronounced in vagotomized dogs than in intact dogs.
(2) Blood ADH titers and MAP were elevated by the occlusion of vertebral arteries in all groups of dogs. However, the elevation of blood ADH titers in denervated dogs was more pronounced than in intact dogs, but less than in vagotomized dogs.
(3) The effects of the occlusion of common carotid arteries on blood ADH titers and MAP were more pronounced than those of the occlusion of vertebral arteries.
These results may suggest that :
a. baroreceptors involved in vasopressin secretion are present in vertebral arteries as well, and that b. the intrathoracic baroreceptors are dominant in controlling vasopressin secretion, while those in common carotid arteries are secondly and those in vertebral arteries thirdly dominant.

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The experiments were performed to investigate the effect of anterior pituitary hormones and sex hormones on the feature of circadian variations in concentrations of norepinephrine (NE) and serotonin (5-HT) in the whole brain, the hippocampus and the hypothalamus of male rats. The effect on spontaneous movement was also examined. Wistar male rats weighing about 180g were housed in a room with constant ambient temperature of 22 ± 1°C under artificial light (light on from 05 : 00 to 19 : 00h).The hypophysectomized rat or the hypophysectomized and castrated rat was administrated subcutaneously with anteropituitary extract (AP extract) every day at 11 : 00h for 5 consecutive days from the day of hypophysectomy. Castration was performed before the day of hypophysectomy. On the day of experiment, these rats were decapitated with a guillotine at 14 : 00, 17 : 00, 20 : 00, 23 : 00, 02 : 00, 05 : 00, 08 : 00, 11 : 00 or 14 : 00h. Concentrations of NE and 5-HT were determined by fluorimetric method. NE was isolated from the whole brain or the hippocampus using Dowx50 cation exchange resin column and from the hypothalamus by alumina batch operation.
The results are as follows :
1) NE concentration in the whole brain revealed a circadian variation, which had its peak at 05 : 00h and was low in the day time, whereas 5-HT concentration showed a fluctuation which had its peak at 14 : 00h in the intact rat. NE and 5-HT contents in the whole brain in the hypophysectomized rat did not show a clear circadian variation. Definite circadian variations of NE and 5-HT contents as observed in the intact rat reappeared in the hypophysectomized rat following the administration of AP extract. Circadian variations of NE and 5-HT contents were not observed in the whole brain of the hypophysectomized and castrated rat, but were restored by the administration of AP extract. However, there was observed a phase shift in these animals.
2) 5-HT content in the hippocampus showed a circadian variation with its peak in the night, at 02 : 00h. This rhythm was not observed in the hypophysectomized rat, but was restored by the administration of AP extract. NE content in the hippocampus of the intact rat showed a circadian variation with a nadir at 05.00h and a peak at 11 : 00h, and this was maintained in the hypophysectomized rat. This pattern was disturbed by the administration of AP extract and the content increased remarkably. NE content in the hippocampus of the hypophysectomized and castrated rat increased also remarkably by the administration of AP extract.
3) NE content in the hypothalamus showed a clean circadian variation with a peak at 20.00h in the intact rat. During the day time the level was relatively low. Similar pattern of circadian variation was still observed in either the hypophysectomized rat or the castrated rat, but there was observed a phase shift of its peak by +180°. Administration of AP extract into the hypophysectomized rat, which had a phase shift by +180°, further advanced the appearance of peak, resulting a phase shift by +270° compared with the time of peak in the intact rat.
4) The spontaneous movement in the hypophysectomized rat, in the castrated rat or the hypophysectomized and castrated rat was remarkably decreased compared with that in the intact rat, but the rhythm similar to that in the intact rat was maintained. The spontaneous movement was-increased following the administration of AP extract.
It is concluded from the present results that the anterior pituitary hormones exert considerable influences on the feature of circadian variations in the brain monoamines levels by themselves and also through the hormones from its target glands. This is supported by the result from the experiment on spontaneous movement.

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The effects of abnormal thyroid function on the hypothalamo-pituitary-adrenal system were studied in 37 hyperthyroid and 16 hypothyroid patients and compared to 38 normal subjects.
1) Plasma corticotropin (ACTH), growth hormone (GH) and cortisol responses to insulin hypoglycemia (0.1 U/kg, iv) were examined. Plasma ACTH was determined by radioimmunoassay (RIA) using two antisera, one directed against the C-terminal portion of ACTH molecule, the other (NPA, 699 FRED) directed against the N-terminal portion. The mean basal ACTH levels determined by C-terminal antibody in normal, hyperthyroid and hypothyroid subjects were 59 ± 11 (SE), 111 ± 35 and 33 ± 20 pg/ml respectively, where a statistically significant difference was not present. However, the maximum levels attained during insulin hypoglycemia were 410 ± 34, 1,379 ± 216 (P<0.01) and 269 ± 58 pg/ml (P<0.05) in the same order. The significant correlation between the maximum ACTH concentration and RT₃U was revealed (r = 0.766, P<0.005). The maximum ACTH levels determined by the N-terminal antibody were also increased in hyperthyroid patients (537± 116 pg/ml, P<0.05), when compared with normal subjects (249 ± 53 pg/ml). By contrast, the plasma GH responses to hypoglycemia were markedly suppressed in both hyper- and hypothyroid patients. The plasma cortisol responses were not significantly different between normal subjects and patients with thyroid dysfunction.
2) t 1/2, V, and MCR of cortisol were obtained from the disappearance curve of intravenously injected 2.5-5 mg of cortisol after overnight dexamethasone suppression, using one pool model. MCR's in normal, hyper- and hypothyroid subjects were 244 ± 22,696 ± 88 (P<0.001) and 129 ± 19 1/24 hrs (P<0.005) respectively.
3) The maximum cortisol production rate (PRmax) was obtained by plasma cortisol levels during 30 and 90 minutes after iv injection of 250 μg of 1-24 ACTH, and t 1/2 and V of cortisol by the formula of Hellman (1970). PRmax's in normal, hyper- and hypothyroidism were 56.7 ± 4.8,102.9 ± 11.6 (P<0.05) and 31.2 ± 2.8 pig/min (P<0.01). The mean plasma cortisol level at 120 minutes after the priming dose-constant infusion of the excess amount of 1-24 ACTH following overnight dexamethasone suppression in hyperthyroidism was significantly less than that of normal subjects (22.6 1.5 vs. 27.3 ± 1.1, P<0.05), while that of hypothyroidism was not significantly different (24.6 ± 0.9 μg/ 100 ml)).
4) The amount of cortisol produced (Pph) and the availability of cortisol secreted in response to a physiological dose (1 μg) of 1· 24 ACTH after overnight dexamethasone suppression were investigated. From the response curve of plasma cortisol, determined by competitive protein binding method, cortisol production (Pph) was calculated by the formula, P = MCR ∫^∞_o C_F dt = MCR × S (S : area under cortisol response curve). Pph's in normal, hyper- and hypothyroid subjects were 3.00 ± 0.15, 3.48 ± 0.56 (N.S.) and 1.88 ± 0.17 mg (P<0.01) respectively. But, the availability of cortisol secreted (area under cortisol concentration curve) after iv injection of 1 μg of 1-24 ACTH in hyperthyroidism was markedly decreased (808 ± 165, P<0.001), while that in hypothyroidism (2,459 ± 313) was not significantly different from normal subjects (1,872 ± 143 min·μg/ 100 ml).

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